RNA modifications play important roles in regulating a wide range of biological processes linked to human health and disease. Several distinct RNA modifications have been identified in human cellular mRNA including base methylations and base isomerization such as the modification from uridine to pseudouridine (?). Emerging evidence suggest that modifications of both RNA genome and transcripts of viral pathogens have a profound impact on viral replication. While there is agreement that several adenosines within HIV are methylated, other RNA modifications have not yet been mapped. In preliminary experiments, we identified several pseudouridines in HIV RNA but the role of these pseudouridines in HIV biology remains poorly understood. We hypothesize that these epitranscriptomic marks on the HIV RNA modulate viral replication and are dependent on the expression of ? writer and ? reader proteins. This proposal will identify the proteins that install and bind to the HIV pseudouridine sites and reveal how ? modifications facilitate HIV replication. Aim 1 will systematically mutate individual ? sites in the HIV genome to identify specific steps in the viral life cycle that are affected by ? modifications. Aim 2 will dissect the HIV-? modified RNA-protein interactome. We will map and validate direct reader proteins for the HIV ? modified sites and examine the functional consequences of these interactions in human cells. Aim 3 will identify the cellular enzymes that install HIV-? modifications, and define their role in the context of HIV infection. We will match specific pseudouridine synthase to individual ? sites in HIV, and perform biological studies upon perturbation of these writer proteins. The proposed studies will define the role of ? in HIV infection and identify the components of the host ? cell machinery necessary and responsible for altering HIV phenotypes in the absence of mutations in the HIV genome. Since changes to the RNA landscape are more malleable than changes at the DNA level, pharmacological targeting of RNA modifications may, in the future, offer novel candidate targets for therapeutic interventions aimed at curbing HIV replication.